Chlorinated cobalt alkyne complexes derived from acetylsalicylic acid as new specific antitumor agents

Article abstract of DOI:10.1039/c7dt04790h

[(Prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS), an organometallic derivative of the irreversible cyclooxygenase-1/2 (COX-1/2) inhibitor acetylsalicylic acid (ASS), demonstrated high growth-inhibitory potential against various tumor cell lines and inhibition of both COX isoenzymes. With the objective of increasing the selectivity for COX-2, we introduced a chlorine substituent in position 3, 4, 5, or 6 of the ASS moiety, respectively. Increased COX-2 selectivity is desirable as this isoenzyme is predominantly related to the development of cancer and abnormal tissue growth. The new compounds were investigated in comprehensive cellular biological assays to identify the impact of the chlorine substitution at the complex on COX-1/2 inhibition, antiproliferative activity, apoptosis, metabolic activity, cell-based COX inhibition, and cellular uptake. Chlorination distinctly reduced the effects at isolated COX-1 (about 25% inhibition at 10 μM; Co-ASS: 82.7%), while those at COX-2 remained almost unchanged (about 65% inhibition at 10 μM; Co-ASS: 78.5%). In cellular systems, with exception of the 6-Cl derivative, all compounds showed notable antitumor activity in COX-1/2 expressing tumor cells (HT-29 (IC₅₀ = 1.5-2.7 μM), MDA-MB-231 (IC₅₀ = 5.2-8.0 μM)), but were distinctly less active in the COX-1/2-negative MCF-7 breast cancer cell line (IC₅₀ = 15.2-22.9 μM). All complexes possess high selectivity for tumor cells, because they did not influence the growth of the non-tumorigenic, human bone marrow stromal cell line HS-5. These findings clearly demonstrate that the interference with the COX-1/2 cascade contributes to the mode of anticancer action of the cobalt alkyne complexes.

Full text of DOI:10.1039/c7dt04790h

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Chlorinated cobalt alkyne complexes derived from
acetylsalicylic acid as new specific antitumor
agents†
Cite this: DOI: 10.1039/c7dt04790h
Victoria Obermoser,‡a Daniel Baecker,‡a Carina Schuster,a Valentin Braun,a
a
Brigitte Kircherb,c and Ronald Gust
*
[(Prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS), an organometallic derivative of the
irreversible cyclooxygenase-1/2 (COX-1/2) inhibitor acetylsalicylic acid (ASS), demonstrated high growth-
inhibitory potential against various tumor cell lines and inhibition of both COX isoenzymes. With the
objective of increasing the selectivity for COX-2, we introduced a chlorine substituent in position 3, 4, 5,
or 6 of the ASS moiety, respectively. Increased COX-2 selectivity is desirable as this isoenzyme is pre-
dominantly related to the development of cancer and abnormal tissue growth. The new compounds were
investigated in comprehensive cellular biological assays to identify the impact of the chlorine substitution
at the complex on COX-1/2 inhibition, antiproliferative activity, apoptosis, metabolic activity, cell-based
COX inhibition, and cellular uptake. Chlorination distinctly reduced the effects at isolated COX-1 (about
25% inhibition at 10 µM; Co-ASS: 82.7%), while those at COX-2 remained almost unchanged (about 65%
inhibition at 10 µM; Co-ASS: 78.5%). In cellular systems, with exception of the 6-Cl derivative, all com-
pounds showed notable antitumor activity in COX-1/2 expressing tumor cells (HT-29 (IC50 = 1.5–2.7 µM),
MDA-MB-231 (IC50 = 5.2–8.0 µM)), but were distinctly less active in the COX-1/2-negative MCF-7 breast
cancer cell line (IC50 = 15.2–22.9 µM). All complexes possess high selectivity for tumor cells, because
they did not influence the growth of the non-tumorigenic, human bone marrow stromal cell line HS-5.
These findings clearly demonstrate that the interference with the COX-1/2 cascade contributes to the
mode of anticancer action of the cobalt alkyne complexes.
Received 19th December 2017,
Accepted 19th February 2018
DOI: 10.1039/c7dt04790h
rsc.li/dalton
PGI2, and thromboxane via their respective synthases. The
constitutively active isoform COX-1 is responsible for basal
Introduction
Cyclooxygenases are homodimeric enzymes catalyzing the PG synthesis in nearly all human tissues. Besides their func-
conversion of arachidonic acid to the cyclic prostaglandins tions in platelet activity, reproduction, and regulation of peri-
(PGs) G2 and H2, which are further converted to PGE2, PGD2, pheral vascular resistance, COX-1-derived PGs are particularly
important in the maintenance of the protecting gastric
mucosa.1,2 The second short-living isoform COX-2 is basally
aDepartment of Pharmaceutical Chemistry, Institute of Pharmacy, Center for
expressed only in the central nervous system, the kidney and
Molecular Biosciences Innsbruck, University of Innsbruck, CCB – Centrum for
in female reproduction organs. However, it is inducible upon
Chemistry and Biomedicine, Innrain 80-82, 6020 Innsbruck, Austria.
stimulation by cytokines, endotoxins, and mitogens in the
E-mail: ronald.gust@uibk.ac.at; Tel: +43-512-507-58200
bImmunobiology and Stem Cell Laboratory, Department of Internal Medicine V
(Hematology and Oncology), Innsbruck Medical University, Anichstrasse 35,
6020 Innsbruck, Austria
context of pathophysiological processes and contributes via
specific
PG
production
to
inflammation
and
carcinogenesis.3–6 COX-2 is upregulated in various types of
cancer, such as colon and breast cancer, osteosarcoma,
chronic myeloid leukemia, melanoma, ovarian, and gastric
cancer.7–10 Its downstream product PGE2 acts as harmful
mediator in cancer tissue. PGE2 inhibits apoptosis, increases
proliferation, initiates angiogenesis, supports invasiveness
and suppresses immune answer via interaction with its
specific receptors EP1–4, leading overall to accelerated tumor
growth and increased tumor aggressiveness.11–14
cTyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck, Austria
†Electronic supplementary information (ESI) available: Synthesis and character-
ization of intermediate products; graphic presentation of crystal violet prolifer-
ation assay (HT-29, MDA-MB-231, MCF-7); graphic presentation of caspase-3/7
induction at concentrations ≤40 µM (HT-29, MDA-MB-231, MCF-7); tabular data
of COX-1/2 inhibition; HPLC chromatograms of stability tests including specifi-
cations of solvent gradient; HPLC chromatograms of purity tests; 1H-NMR
spectra, mass spectra and HPLC chromatograms of final products; graphite
furnace time–temperature program. See DOI: 10.1039/c7dt04790h
‡These authors contributed equally to the publication.
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The cancer chemopreventative and adjuvant chemothera-
Acetylation of the chlorine-substituted salicylic acids with
peutic properties of COX inhibitory nonsteroidal anti-inflam- acetic anhydride and triethylamine (TEA) in tetrahydrofuran
matory drugs (NSAIDs) have therefore been extensively (THF) resulted in the respective chlorine-substituted acetyl-
explored in cancers expressing the enzyme. Acetylsalicylic acid salicylic acids (3/4/5/6-Cl-ASS), which were subsequently
(ASS) exhibited good results in the prevention of both breast esterified with propargyl alcohol (leading to propargyl-3/4/5/
and colon cancers or as adjuvant in combination therapy.15–18 6-Cl-ASS) according to
a
method described by Steglich
But to the best of our knowledge it does not cause any cyto- et al. using N,N′-dicyclohexylcarbodiimide (DCC) and
toxic effect in vitro or solely administered in vivo.
the Steglich-catalyst 4-(dimethylamino)pyridine (DMAP).26
In our group, we tried to increase the antitumor activity of Propargylchlorosalicylates, which were built as by-products in
ASS by coordinating it to an organometallic moiety. For this this reaction were reacetylated as described above without sep-
purpose, ASS was esterified with propargyl alcohol and bound aration from the respective ASS ester (Scheme 1).
to dicobalthexacarbonyl.19 The resulting [(prop-2-ynyl)-2-acet-
Finally, reaction of the propargyl-3/4/5/6-chloroacetylsalicy-
oxybenzoate]dicobalthexacarbonyl (Co-ASS) showed consider- lates with dicobaltoctacarbonyl in dry THF under an argon-
able growth inhibitory potential in colon (HT-29) and breast modified atmosphere yielded the respective dicobalthexacarbo-
(MDA-MB-231) cancer cell lines with IC50 values in the low nyl complexes (Scheme 1).
micromolar range. Compared to ASS, the potency to inhibit
COX isoenzymes 10-fold increased for both COX-1 and COX-2,
Biological evaluation
suggesting an interference with the arachidonic acid cascade
COX-1/2 isoenzyme inhibition. ASS was used as a positive
as a probable mode of action.20 However, the high inhibitory control and tested for COX-1/2 inhibition. At 10 µM only a
effect on COX-1 points to therapy-limiting side effects, such as marginal reduction of enzymatic activity was determined at
ulcers, which can be reduced by shifting the COX isoenzyme COX-1 (28.8%), while it was completely inactive at COX-2
selectivity towards COX-2.21
(0.6%). In a 10-fold higher concentration (100 µM), the inhibi-
The amino acids Ile523, His513, and Ile434 in COX-1 lead tory properties increased to 73.3% (COX-1) and 59.4% (COX-2)
to a smaller binding cave compared to COX-2 (Val523, Arg513, (Table 1). These results go along with results that have been
and Val434). Especially, Ile523 in COX-1 blocks the access to a published previously by our group.20,27
small hydrophilic side pocket. The larger substrate channel in
COX-2 was already utilized to design COX-2 selective inhibition of ASS to about 30% (100 µM) at both isoenzymes,
NSAIDs.1,22–24
while substitution at position 4 or 5 maintained the inhibitory
A chlorine substituent at position 3 or 6 reduced the COX
Therefore, we tried to use these differences in the volume effects nearly unchanged (see Table S1 in ESI†). After esterifi-
of the binding pocket to reduce the COX-1 inhibitory effects cation of the Cl-ASS moiety with propargyl alcohol, the activity
and to maintain the activity at COX-2 by introduction of a was about 30% at COX-1 and about 50% at COX-2, indepen-
bulkier chlorine substituent into the aromatic moiety of dent of the position of the chlorine substituent at ASS. The
Co-ASS. The derivatives 3-Cl-Co-ASS, 4-Cl-Co-ASS, 5-Cl-Co-ASS, COX-2 inhibition induced by the unsubstituted propargyl-ASS
and 6-Cl-Co-ASS were investigated for COX inhibitory effects at was slightly weaker (34.5%). Binding of propargyl-ASS to di-
isolated or human recombinant isoenzymes and in cell culture cobalthexacarbonyl, resulting in Co-ASS, distinctly increased the
experiments against HT-29, MDA-MB-231, and MCF-7 cells COX inhibitory effects. An inhibition of about 80% was deter-
regarding their influence on proliferation, metabolic activity, mined at both isoenzymes at 10 µM. Interestingly, the activity
and caspase-3/7 induction. Furthermore, we studied their of the chlorine-substituted propargyl-ASS derivatives at COX-1
potential to inhibit intracellular PGE2 production in HT-29 cannot be increased upon coordination. At COX-2, however, all
cells and the time-dependent cellular uptake was monitored to chlorinated Co-ASS derivatives showed the same effects as the
complete the characterization of their pharmacological pro- parent compound Co-ASS. This finding clearly documents that
files. Finally, the selectivity of the compounds towards cancer chlorine substituents reduced the unwanted COX-1 inhibition
cells was investigated using the healthy (non-cancerous) and shifted therefore the selectivity to COX-2.
human bone marrow stromal cell line HS-5 for comparison.
COX-1/2 expression in tumor cells. For the interpretation of
the results and the discussion about the participation of COX
inhibition in the mode of action, all cell lines used for biologi-
cal investigations within this project were characterized for
COX-1 and COX-2 expression levels with TaqMan® quantitative
real time polymerase chain reaction (qPCR). From the Cq value
Results and discussion
Synthesis
6-Chlorosalicylic acid (6-Cl-SS) was obtained by reacting of each sample, which is calculated from the point at which
2-chloro-6-fluorobenzoic acid with powdered sodium hydrox- the curvature of the amplification curve is maximal, an absol-
ide in dimethyl sulfoxide (DMSO). Thereby, the fluorine substi- ute value of copy numbers was calculated using a standard
tuent was selectively exchanged by
a hydroxyl group curve. Before calculation, all values were normalized to the
(Scheme 1).25 3-Chlorosalicylic acid (3-Cl-SS), 4-chlorosalicylic housekeeper gene β-actin. The results are depicted in Fig. 1.
acid (4-Cl-SS), and 5-chlorosalicylic acid (5-Cl-SS) were com-
mercially available.
The MDA-MB-231 (breast cancer) and the HT-29 (colon car-
cinoma) cell lines express both COX-1 and COX-2, though at
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Scheme 1 Synthesis of the propargyl-3/4/5/6-chloroacetylsalicylates and related dicobalthexacarbonyls. Ar, argon; DCC, N,N’-dicyclohexyl-
carbodiimide; DCM, dichloromethane; DMAP, 4-(dimethylamino)pyridine; DMSO, dimethyl sulfoxide; RT, room temperature; TEA, triethylamine;
THF, tetrahydrofuran.
Table 1 Inhibition of isolated ovine COX-1 and human recombinant
COX-2 isoenzymes
COX-1 inhibitiona
[%]
COX-2 inhibitiona
[%]
Compound
ASS [10 µM]
ASS [100 µM]
28.8 3.7
73.3 8.7
25.4 4.4
27.0 6.1
32.5 1.9
82.7 3.2
24.9 2.6
22.9 1.7
25.0 1.0
24.4 3.0
0.6 2.8
59.4 8.7
34.5 4.2
29.5 3.5
52.2 5.7
78.5 6.1
71.4 8.3
69.9 2.7
64.0 7.5
70.5 3.5
Propargyl-ASS [100 µM]
3-Cl-ASS [100 µM]
Propargyl-3-Cl-ASS [100 µM]
Co-ASS [10 µM]
3-Cl-Co-ASS [10 µM]
4-Cl-Co-ASS [10 µM]
5-Cl-Co-ASS [10 µM]
6-Cl-Co-ASS [10 µM]
a Values are represented as % COX inhibition calculated from initial
activity of vehicle treated enzyme. Each experiment was conducted ≥3
times independently and the values are presented as mean SD.
■
Fig. 1 COX-1 ( ) and COX-2 ( ) expression of the cell lines HT-29,
MDA-MB-231, and MCF-7. Quantification was performed with
TaqMan® qPCR using β-actin as internal normalization standard.
a
different levels. The highest COX-1/2 expression was found in
HT-29 cells. In MDA-MB-231 cells, total copy numbers per µg
Absolute values were calculated with the standard curve method and
are only the half compared to HT-29 cells. This is the conse- represent the mean SD of 3 independent experiments.
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Table 2 Inhibition of cell growth determined in a crystal violet cell
biomass assay
cells (highest concentration tested: 40 µM) with IC50 values
ranging from 15.2 to 22.9 µM. This finding is an indication
but not the proof that COX-1 and COX-2 inhibition are
involved in the mode of action. However, the 6-Cl-substituted
compound (IC50 = 41.2 µM) displayed a far smaller effect in
comparison to the other derivatives.
The determined effects against COX-negative MCF-7 cells
demonstrate the interference in further pathways in the cells.
Therefore, the growth inhibitory potency cannot be discussed
in relation to the COX-1/2 inhibition only. Further parameters
must be taken into consideration. This is especially true for
6-Cl-Co-ASS, which was the complex with the lowest antiproli-
ferative effect, while being equally potent as the other com-
plexes in the COX-1/2 assay at isolated enzymes.
IC50 [µM]
Compound
HT-29
MDA-MB-231
MCF-7
ASS
≥100
≥100
≥100
Propargyl-ASS
3-Cl-Co-ASS
4-Cl-Co-ASS
5-Cl-Co-ASS
6-Cl-Co-ASS
Co-ASS
≥100
≥100
≥100
2.67 0.47
1.51 0.12
2.04 0.11
21.1 4.0
1.15 0.10
3.52 0.48
8.04 0.30
5.24 0.33
7.85 0.93
22.4 1.7
10.1 0.6
3.57 1.06
22.9 1.9
15.2 1.3
20.7 2.9
41.2 3.2
16.4 1.4
4.02 1.11
Cisplatin
IC50 values are calculated as the concentration where 50% of the com-
pounds maximum antiproliferative activity compared to the vehicle
treated control were achieved and are presented as mean SD of ≥4
independent experiments.
Stability studies. For Co-ASS a possible mode of action
includes acetylation of the COX enzymes.20 If the acetyl group
is cleaved in aqueous solution before entering the cells, the
resulting salicylic acid derivative might be less active. The
quence of a distinctly reduced COX-1 level. The expression of incubation time in the cell-based crystal violet assay is 72 h
COX-2 is comparable in both cell lines. Interestingly, the and allows a possible deacetylation of the complexes, probably
MCF-7 cell line used in this study is negative for both, COX-1 influenced by the chlorine substituent at positions 3, 4, 5, or
and COX-2, and allows therefore the monitoring of possible 6. Decomposition in the COX assay is not very likely because of
COX independent cytotoxic effects.
an incubation time of only 10 min.
Antiproliferative effects. The antiproliferative activity of the
In order to study the influence of the chlorine substituents
newly synthesized compounds was analyzed in vitro using an on the stability in aqueous solution, the ASS derivatives and
already published crystal violet assay.28 In this assay the cell the ASS-propargyl esters were dissolved in 30% DMSO/0.1 M
mass reduced by the compounds is measured as parameter.
phosphate buffer (pH 7.4) to a concentration of 0.5 mg mL−1
The precursors (ASS and propargyl-ASS derivatives), the and analyzed time-dependently for degradation at 37 °C by
chlorine-substituted complexes, and Co-ASS were screened HPLC analysis. This mixture was used to guarantee that educts
against the HT-29, MDA-MB-231, and MCF-7 cell lines. and all degradation products remain in solution.
Cisplatin was used as positive control. The IC50 values pre- Unfortunately, the solubility of the respective cobalt complexes
sented in Table 2 were determined after an incubation period was not high enough to study them under in vitro conditions.
of 72 h. Concentration–activity curves are submitted as ESI.†
The position of the chlorine substituent at the aromatic
As expected, ASS and propargyl-ASS were inactive up to a con- moiety strongly influenced the deacetylation. If the substituent
centration of 100 µM. All precursors were excluded from the is located at position 4 or 5, the acetylsalicylic ester degraded
extended studies on metabolic activity and apoptosis because into the salicylic acid (SS) with t1/2 = 8.5 h and 12.5 h,
chlorination of the ASS and the propargyl-ASS moiety did not respectively.
increase the antiproliferative effects.
Cisplatin showed comparable activity at all cell lines with stable (t1/2 (3-Cl-ASS) = 32.5 h; t1/2 (6-Cl-ASS) = 41.7 h).
IC50 values of about 3.5–4.0 µM.
If the propargyl esters were comparably incubated, ASS,
The 3-chloro and 6-chloro derivatives were much more
The activity of the dicobaltcarbonyl complexes correlates SS, and the SS-propargyl ester were identified as degradation
with the total COX level in the cells: the higher the COX level products. The stability increased about 1.5-fold upon esterifi-
the higher the sensitivity of cell against the compounds. For cation to the propargyl ester in the case of the 3-, 4-, and 5-Cl-
instance, the lead compound Co-ASS was highly active against substituted compounds (t1/2 (propargyl-3-Cl-ASS) = 53.1 h;
HT-29 cells (IC50
= 1.15 µM) but less active against t1/2 (propargyl-4-Cl-ASS) = 13.5 h; t1/2 (propargyl-5-Cl-ASS) =
MDA-MB-231 (IC50 = 10.1 µM) and especially MCF-7 (IC50
16.4 µM) cells.
=
19.0 h). A very contrary result was obtained after conden-
sation of 6-Cl-ASS with propargyl alcohol. The half-life
After chlorination of the ASS moiety in position 3, 4 or 5, decreased from 41.7 h to 7.9 h. After 48 h the 6-ASS propargyl
the effects on the HT-29 cell line remained nearly unchanged ester was completely hydrolyzed. In contrast to the other
(IC50 = 1.5–2.7 µM) and slightly decreased against MDA-MB-231 derivatives, the hydrolysis of the propargyl ester was preferred
cells in the order Co-ASS (IC50 = 10.1 µM) < 3-Cl-Co-ASS (IC50
8.04 µM) ≈ 5-Cl-Co-ASS (IC50 = 7.85 µM) < 4-Cl-Co-ASS (IC50
=
=
and the cleavage of the acetyl group played a subordinate
role.
5.24 µM). In contrast, the 6-chloro derivative was considerably
less potent with IC50 ≈22 µM at both cell lines.
Although 6-Cl-Co-ASS could not be investigated under com-
parable conditions, it can be assumed that it is hydrolyzed
The 3-, 4-, and 5-Cl-substituted complexes and also Co-ASS into 6-Cl-ASS and propargyl-Co2(CO)6 to a higher extent prior
showed distinctly weaker antiproliferative effects in MCF-7 to uptake into cells compared to all other compounds.
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The high-performance liquid chromatography (HPLC) chro- 6-Cl-Co-ASS (Fig. 2b). As against HT-29 cells, 6-Cl-Co-ASS was
matograms are submitted as ESI.† inactive. 5-Cl-Co-ASS as most active complex reduced the meta-
Metabolic activity. The metabolic activity of the cells treated bolic activity to 60% (20 µM) and 10% (40 µM) documenting
with the cobalt complexes was assessed in a modified MTT that MDA-MB-231 cells are slightly less sensitive to the chlo-
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) rine-substituted compounds.
colorimetric assay. The capability of NAD(P)H-dependent cellu-
Interestingly, the metabolic activity of the MCF-7 cells was
lar oxidoreductase enzymes, which correlates with the number not significantly affected by any cobalt complex (Fig. 2c), not
of viable cells, is determined via reduction of the tetrazolium even at the highest concentration (40 µM) used.
dye MTT to its insoluble, purple colored formazan. Hence, this
assay discriminates between living and dead/apoptotic/necro- MB-231) were more sensitive against the cobalt complexes
tic cells. than the cell line lacking COX-1/2 (MCF-7). It should be noted,
In summary, cells expressing COX-1/2 (HT-29, MDA-
In HT-29 cells, 4-Cl-Co-ASS, and Co-ASS reduced the meta- that the metabolic activity is reduced in all cell lines only at
bolic activity to approximately 50% at 20 µM. 5-Cl-Co-ASS was relatively high concentrations, which were far above the IC50
more active and reduced the metabolic activity at 20 µM to value determined in the proliferation assay. These findings
24% and at 40 µM to 6%, while the 3-chloro derivative suggest that the cobalt complexes have a cytostatic effect on
decreased the activity at these concentrations only to 69% the cells already at low concentrations without interfering with
(20 µM) and 27% (40 µM), respectively. 6-Cl-Co-ASS was com- their mitochondrial metabolic turnover.
pletely inactive (Fig. 2a).
Induction of apoptosis. The capacity of the compounds to
In MDA-MB-231 cells nearly the same tendency was induce programmed cell death was determined in a caspase-3/7
observed: 5-Cl-Co-ASS > Co-ASS ≈ 3-Cl-Co-ASS > 4-Cl-Co-ASS > assay. Both caspases are apoptosis-related cysteine peptidases
activated via external or intrinsic signalling pathways with the
objective of degrading cell components. In contrast to necro-
sis, which is a pathophysiological event, apoptosis is the phys-
iological destruction of cells, which might be harmful for the
organism.
A significant induction of caspase-3/7 was only observed at
a concentration of 40 µM, why only these data are presented in
Fig. 3. Further data are available as ESI.†
In accordance with the results of the MTT assay, in MCF-7
cells the cobalt complexes only marginally induced caspase-3/
7-related apoptosis. In MDA-MB-231 and HT-29 cells, 3-, 4-,
and 5-Cl-Co-ASS as well as Co-ASS increased the caspase-3/7
content 3- to 5-fold independent on the position of the chlor-
ine substituent.
In general, the caspase-3/7-inductive effect was more pro-
nounced in HT-29 cells compared to the MDA-MB-231 cell
line. These data correlate with the results of the MTT assay.
It is obvious that Co-ASS and its chlorine derivatives show
cytostatic activity at concentrations <20 µM as the cells neither
lose their metabolic capacity nor migrate to an apoptotic state.
Fig. 2 Effect of cobalt complexes on metabolic activity after 72 h
determined in a MTT assay in (a) HT-29, (b) MDA-MB-231, and (c) MCF-7
Fig. 3 Induction of caspase-3/7 in HT-29, MDA-MB-231, and MCF-7
cell lines treated with the cobalt complexes. Values represent the mean
SD of ≥4 independent assays. Asterisks represent statistical signifi-
cance (p < 0.05 vs. controls).
cells. Values represent the mean
SD of ≥4 independent assays.
Asterisks represent statistical significance (p < 0.05 vs. controls).
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PGE2 inhibition. HT-29 cells were selected for investigations
of the cellular COX inhibition. These cells express the
highest amounts of COX-1/2 and thus represent an established
model for the determination of intracellular PGE2 synthesis
via EIA.
COX-1/2 catalyze the double oxygenation of added arachido-
nic acid to PGG2 and the subsequent reduction to PGH2 by its
peroxidase (POX) active site. PGH2 is then converted to PGE2
by tissue- and cell-specific isomerases. PGE2 is relatively stable
upon release of the cell to the medium and is therefore a good
analyte to quantify the arachidonic acid turnover by COX-1/2.
Results of this assay are presented in Fig. 4.
Treatment of HT-29 cells with ASS reduced the intracellular
PGE2 synthesis (25.4%) only at 100 µM. The respective propar-
gyl ester (ASS-propargyl) caused marginal PGE2 inhibition
(18%) at 10 µM. Coordination to Co2(CO)6 led to the lead struc-
ture Co-ASS, which is the most active compound in this study
(57.5% at 10 µM). Chlorine substituents at the aromatic
moiety slightly decreased the influence on the PGE2 release if
they are in position 3 (37.7%), 4 (48.5%), or 5 (41.9%). 6-Cl-Co-
ASS reduced the PGE2 level only by 18%. These findings corre-
late well with the results obtained in the other tests against
this cell line and clearly point to a participation of COX inhi-
bition in the mode of action. It is worthy to mention that PGE2
inhibition distinctly higher than 20% is necessary to influence
the cell growth because ASS as well ASS-propargyl were inactive
and 6-Cl-Co-ASS was only marginally active at the used
concentrations.
Cellular uptake. The uptake of a drug into the target cell is a
crucial parameter for its activity as the intracellular concen-
tration influences the extent of interaction with the drug
targets. To assess the cellular uptake of the alkyne complexes
Fig. 5 Determination of cellular uptake (pmol complex per µg protein)
into (a) HT-29, (b) MDA-MB-231, and (c) MCF-7 after 1, 2, 4, 6, and 24 h
(concentration 10 µM), the cobalt content in HT-29, MCF-7, of incubation with 3-Cl-Co-ASS ( ), 4-Cl-Co-ASS ( ), 5-Cl-Co-ASS ( ),
◆
6-Cl-Co-ASS ( ), Co-ASS ( ) at a concentration of 10 µM. Data points
and MDA-MB-231 cells after set incubation periods was quanti-
fied by high-resolution continuum-source atomic absorption
spectrometry (HR CS AAS). Results are graphically depicted in
Fig. 5.
represent mean of 3 independent experiments SE.
Cellular uptake into HT-29 cells. All complexes showed a fast
cellular uptake with a maximum after 4–6 h. While the level
remained nearly constant for 5-Cl-Co-ASS and 6-Cl-Co-ASS, it
slowly decreased for Co-ASS, 3-Cl-Co-ASS as well as 4-Cl-Co-ASS
after 6 h of incubation. After 24 h, with the exception of 6-Cl-
Co-ASS all complexes showed nearly the same cobalt content
of about 4.5 pmol µg−1 protein. For 6-Cl-Co-ASS only the half
amount was determined (2.2 pmol μg−1) and could be the
explanation of the lesser PGE2 level measured in the above
mentioned PGE2 assay.
Cellular uptake into MDA-MB-231 cells. In MDA-MB-231 cells
the complexes were taken up to a lesser extent compared to
HT-29 cells during the first 6 h of incubation. In case of Co-
ASS and 6-Cl-Co-ASS the maximum was reached and the cobalt
content did not change during the following 18 h of incu-
bation. After 24 h, 4.5 pmol µg−1 (Co-ASS) and 1.5 pmol µg−1
(6-Cl-Co-ASS) were determined. Interestingly, the cobalt
amount further increased for 3-Cl-Co-ASS, 4-Cl-Co-ASS, and
Fig. 4 Inhibition of intracellular PGE2 synthesis assessed in HT-29
colon carcinoma cells treated with ASS and cobalt complexes at 10 µM.
Values represent the mean SD of 3 independent assays.
5-Cl-Co-ASS to 6.0 pmol µg−1, 6.2 pmol µg−1, and 7.2 pmol µg−1
,
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Co-ASS are less than 50% of its derivatives (45-fold, 18-fold,
and 27-fold, respectively).
Metabolic activity in the human bone marrow stromal cell
line HS-5. The effects of the Co-ASS derivatives against non-
cancerous cells were studied exemplarily against the HS-5 cell
line. As depicted in Fig. 7, all complexes did not influence the
metabolic activity of the HS-5 cells, which points to a selective
damage of COX-positive tumor cells (compare Fig. 2 and 7).
Conclusions
Fig. 6 Maximal accumulation factors of the complexes (10 µM)
exposed to HT-29, MDA-MB-231, and MCF-7 cells. Values represent the
mean SD of 3 independent experiments.
In continuation of our previously published study about the
unselective COX-1/2 inhibitor Co-ASS, we synthesized its 3/4/5/
6-chlorine-substituted derivatives. The aim of this study was to
increase the selectivity for the COX-2 isoenzyme. The chlorine
substituents at the Co-ASS should utilize on the one hand the
larger binding cave of COX-2 to promote activity at this iso-
enzyme and the other hand they should reduce the binding to
COX-1 due to steric repulsion. Indeed, all complexes had high
inhibitory potency against isolated COX-2 (60–80% inhibition
at 10 µM) and low effects at COX-1 (about 20% at 10 µM).
Biological effects (inhibition of proliferation and metabolic
activity, induction of apoptosis) were only determined in cells
with COX expression. Especially the inactivity against COX-
negative MCF-7 cell points to an interference in the arachido-
nic acid oxidation cascade as part of the mode of action. The
missing effects of 6-Cl-Co-ASS in cellular systems can be
explained by its low stability in aqueous solution and/or by a
distinctly reduced cellular uptake. Co-ASS and its Cl-substi-
tuted derivatives demonstrated high selectivity against tumor
cells, because they were completely inactive at the healthy,
non-tumorigenic human bone marrow stromal cell line HS-5.
respectively. Saturation could not be observed. After a fast
initial uptake within the first 2 h, 3-/4-/5-Cl-Co-ASS displayed
nearly linear uptake kinetics expressed by their coefficients of
determinations (r2). The values of r2 are 0.9696, 0.9982, and
0.9934, respectively.
Cellular uptake into MCF-7 cells. Co-ASS and 3/4/5-Cl-Co-ASS
revealed in MCF-7 cells comparable uptake profiles. They
reached after 6 h cobalt levels of 3.7–5.1 pmol µg−1, which
further increased until the end of the experiment (24 h). After
24 h, for 3-Cl-Co-ASS an intracellular amount of 6.2 pmol μg−1
and 4-Cl-Co-ASS of 5.5 pmol μg−1 were measured. The highest
uptake showed 5-Cl-Co-ASS with 6.4 pmol μg−1 after 24 h,
slightly higher than Co-ASS (6.0 pmol µg−1). Again, the lowest
level was determined for 6-Cl-Co-ASS. Interestingly, it reached
the highest level of 2.0 pmol μg−1 after 2 h that decreased
during the following incubation period to 1.5 pmol μg−1
.
If the protein contents are correlated with the cell volumes,
the accumulation grades can be calculated.29,30 Compared to
the extracellular concentration of 10 µM, the complexes were
strongly accumulated in the tumor cells (Fig. 6). The highest
accumulation grades were measured within 24 h for 3-/4-/5-Cl-
Co-ASS in HT-29 cells (90- to 110-fold), while MDA-MB-231 and
MCF-7 cells accumulated the complexes to a lower extent (64-
to 77-fold and 62- to 73-fold, respectively). The values of 6-Cl-
Experimental section
General material and methods
All educts, solvents, and other chemicals were purchased from
Sigma-Aldrich, TCI Chemicals, Fluka, or Alfa Aesar. Solvents
were purchased in appropriate purity or distilled prior to utiliz-
ation. Milli-Q water was obtained from a Milli-Q Gradient A10
water purification system (Merck Millipore, Billerica, USA).
Column chromatography was performed using silica gel 60
(particle size 40–63 µm). Thin layer chromatography (TLC) was
carried out using Polygram® SIL G/UV254 polyester foils with
a 0.2 mm layer of silica gel with fluorescence indicator
(Macherey-Nagel, Düren, Germany). Detection was performed
by observation under UV-light at 254 and/or 365 nm. Melting
points were measured with a Kofler heatable microscope.
1H-Nuclear magnetic resonance spectra (1H-NMR-spectra) were
recorded using a Gemini 2000 NMR Spectrometer (Varian,
Palo Alto, USA, now part of Agilent Technologies, Santa Clara,
USA) at 200 MHz in DMSO-d6 with tetramethylsilane as
internal standard. Chemical shifts are given in parts per
million (ppm). Coupling constants are given in Hertz (Hz).
Fig. 7 Effect of cobalt complexes on metabolic activity after 72 h
determined in a MTT assay in HS-5 cells. Values represent the mean
SD of ≥4 independent assays. Asterisks represent statistical significance
(p < 0.05 vs. controls).
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High resolution mass spectrometry (HR-MS) was performed 6.12 (s, 1H, CH), 5.47 (s, 1H, CH2), 2.35 (s, 3H, CH3);
using an Orbitrap Elite (Thermo Fisher Scientific, Waltham, IR (ν cm−1): 3128 w (Aryl-C–H), 2962 w, 2928 w, 2853 w (C–H),
USA). Infrared (IR) spectra were recorded using an Alpha FT-IR 2094 m, 2026 s, 2003 s (CuC), 1762 s, 1730 s (CvO), 1603 w,
Spektrometer (Bruker, Billerica, USA). HPLC measurements 1579 w (CvC), 1369 m (δ –CH3), 1188 s, 1069 s (C–O); HR-MS:
were conducted with an instrument from Shimadzu (Duisburg, m/z calculated for C18H9ClCo2O10 [2M + Na]+: 1098.6892,
Germany) equipped with an autosampler SIL-20A HT, a pump found: 1098.6980; HPLC [MeOH/water (70 : 30)]: purity 99.6%.
LC20 AD, a column oven CT0-10AS VP and a diode-array detec-
[2-Acetoxy-6-chloropropargylbenzoate]dicobalthexacarbonyl
tor SPD-M20A with deuterium and tungsten lamp. (6-Cl-Co-ASS). Red crystals; yield: 0.053 g (0.098 mmol, 50%);
a
Separation was performed over an Eurospher 100-5 C18 (250 × mp >150 °C. 1H-NMR (200 MHz, CDCl3): δ 7.39 (dd, 3J =
4 mm) column (Knauer, Berlin, Germany). Purity of all final 8.4 Hz, 1H, ArH-4), 7.33 (d, 1H, ArH-5), 7.12 (d, 3J = 8.0 Hz, 1H,
products was above 95%. Absorbance and luminescence in ArH-3), 6.11 (s, 1H, CH), 5.23 (s, 1H, CH2), 2.28 (s, 3H, CH3);
biological assays were measured with an Enspire multimodal IR (ν cm−1): 3094 w (Aryl-C–H), 2959 w, 2923 w, 2851 w (C–H),
plate reader (PerkinElmer Life Sciences, Waltham, USA).
2098 m, 2054 s, 2004 s (CuC), 1774 s, 1736 s (CvO), 1596 m
(CvC), 1369 m (δ –CH3), 1259 s, 1183 m, 1100 s (C–O);
HR-MS: m/z calculated for C18H9ClCo2O10 [2M
Chemistry
+
Na]+:
General procedure for the synthesis of alkinyl cobalt com- 1098.6892, found: 1098.6980; HPLC [MeOH/water (70 : 30)]:
plexes. 50 mg (0.2 mmol) of propargyl-Cl-ASS was dissolved in purity 100%.
anhydrous THF and put into a three-necked-flask, equipped
with an argon connection, a condenser with a bubble counter
and a plug. 71 mg (0.21 mmol, 1.05 equivalent) of dicobal-
toctacarbonyl were quickly weighed in a dry weighing dish and
added to the reaction mixture, which was subsequently stirred
for 4 h at room temperature. The reaction solution turned to
reddish and evolution of carbon monoxide was observed
during the first minutes. Reaction was stopped by adding
approximately 1 g of silica gel. The solvent was evaporated
under reduced pressure. Purification was carried out by
column chromatography using silica gel, with petroleum/ethyl
acetate (9 : 1) as eluent. The coloured fractions were collected.
The first fraction contained the alkinyl cobalt complex of the
salicylic acid derivative and the second one contained the
chloroacetylsalicylic acid ester cobalt complex.
Stability studies
The 0.1 M phosphate buffer was prepared by diluting 2.26 mL
of a NaH2PO4·H2O solution (13.8 g NaH2PO4·H2O in 100 mL of
water) and 15.48 mL of a Na2HPO4 solution (8.9 g Na2HPO4 in
100 mL of water) with water to a final volume of 100 mL. 5 mg
of the test substance were weighed in a 10 mL volumetric
flask, dissolved in 3 mL of DMSO and filled up with 0.1 M
phosphate buffer (pH 7.4) to the mark. A solution of 4.01 mg
5-chlorosalicylic acid dissolved in 3 mL of DMSO was filled up
to 10 mL with phosphate buffer and was used as reference
solution.
The mobile phase was a two-component solvent (aceto-
nitrile and acetic acid 1%), which was run in a gradient. The
gradient specifications can be found in Table S3 of the ESI.†
[2-Acetoxy-3-chloropropargylbenzoate]dicobalthexacarbonyl
(3-Cl-Co-ASS). Red crystals; yield: 0.052 g (0.096 mmol, 48.8%);
mp >150 °C. 1H-NMR (200 MHz, CDCl3): δ 7.98 (d, 1H, ArH-6),
7.64 (d, 1H, ArH-4), 7.22 (dd, 1H, ArH-5), 6.12 (s, 1H, CH), 5.48
Biology
General cell culture methods. The colon cancer cell line
(s, 1H, CH2), 2.41 (s, 3H, CH3); IR (ν cm−1): 3091 w (Aryl-C–H), HT-29, the hormone-independent breast cancer cell line
2917 w, 2852 w (C–H), 2094 m, 2012 s, 2001 s (CuC), 1775 s, MDA-MB-231, and the hormone-sensitive breast cancer cell
1719 s (CvO), 1595 m, 1572 w (CvC), 1364 m (δ –CH3), 1251 line MCF-7 were obtained from the cell line service (CLS,
s, 1183 s, 1105
C18H9ClCo2O10 [2M + Na]+: 1098.6892, found: 1098.6979; line HS-5 was kindly provided by Dr Karin Jöhrer, Tyrolean
HPLC [MeOH/water (70 : 30)]: purity 99.7%. Cancer Research Institute. HT-29, MDA-MB-231, and MCF-7
s (C–O); HR-MS: m/z calculated for Eppelheim, Germany). The human bone marrow stromal cell
[2-Acetoxy-4-chloropropargylbenzoate]dicobalthexacarbonyl cells were maintained as monolayer cultures in Dulbecco’s
(4-Cl-Co-ASS). Red crystals; yield: 0.050 g (0.093 mmol, 50%); modified eagle medium (DMEM) without phenol red, with
1
mp >150 °C. H-NMR (200 MHz, CDCl3): δ 8.04 (s, 1H, ArH-6), glucose (4.5 g L−1) (GE Healthcare), supplemented with fetal
7.28 (s, 2H, ArH-3, ArH-5), 6.12 (s, 1H, CH), 5.47 (s, 1H, CH2), calf serum (FCS; 10%; Biochrom) and L-glutamine (584 mg L−1
,
2.36 (s, 3H, CH3); IR (ν cm−1): 3092 w (Aryl-C–H), 2925 w, 2852 GE Healthcare). The cells were cultivated in a humidified
w (C–H), 2097 m, 2053 s, 2006 s (CuC), 1775 s, 1725 s (CvO), atmosphere (5% CO2/95% air) at 37 °C and passaged twice a
1597 s, 1568 (CvC), 1369 m (δ –CH3), 1184 s, 1092 s (C–O); week. HS-5 cells were grown in RPMI 1640 without phenol
HR-MS: m/z calculated for C18H9ClCo2O10 [2M
1098.6892, found: 1098.6977; HPLC [MeOH/water (70 : 30)]: glutamine (2 mM), penicillin (100 U mL−1), streptomycin
purity 95.6%.
(100 µg mL−1) and fetal bovine serum (FBS; 10%; all from
+
Na]+: red (PAA Laboratories, Pasching, Austria), supplemented with
[2-Acetoxy-5-chloropropargylbenzoate]dicobalthexacarbonyl Invitrogen Corporation, Gibco, Paisley, Scotland) at 37 °C in a
(5-Cl-Co-ASS). Red crystals; yield: 0.065 g (0.121 mmol, 61%); 5% CO2/95% air atmosphere and fed twice weekly. Cell lines
1
mp >150 °C. H-NMR (200 MHz, CDCl3): δ 8.08 (s, 1H, ArH-6), were authenticated by typing short tandem repeats and rou-
7.54 (d, 1H, 3J = 7.8 Hz, ArH-4), 7.08 (d, 3J = 8.0 Hz, 1H, ArH-3), tinely monitored for mycoplasma infection.
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Analysis of relative COX-1 and COX-2 expression. COX-1 and to the manufacturer’s instructions. To exclude unspecific
COX-2 expression in tested tumor cell lines was determined via staining by FCS-containing medium, the optical density of the
qPCR. Total RNA was isolated with a RNA isolation kit (RNeasy respective medium was subtracted. Metabolic activity in the
mini Kit) using a QIAshredder for homogenization of the cell absence of the compound was set at 100%.
lysate (both Qiagen, Hilden, Germany) according to the manu-
facturer’s protocol. The synthesis of cDNA was performed growing cells were seeded in 96-well plates (5 × 104 cells per
using High-Capacity cDNA Reverse Transcription Kit well, 50 µL per well) in triplicates. The compounds were added
Determination of caspase-3/7 induction. Exponentially
a
(Applied Biosystems, Foster City, USA). Quantification of cDNA in appropriate concentrations after 3 h. After cultivation for
was conducted with a qPCR Taqman Assay using a FAMTM- 24 h at 37 °C in a 5% CO2/95% air atmosphere, supernatant
labeled probe (Applied Biosystems) according to the manufac- was transferred to white 96-well plates and induction of apop-
turer’s protocol. Amplification was performed with
a
tosis was measured by caspase-3/7 detection using a Caspase-
Lightcycler®96 (Roche Diagnostics, Basel, Switzerland). The 3/7 Glo® Kit (Promega, Madison, USA) according to the manu-
housekeeper gene β-actin was quantified as loading control facturer’s instructions. Values were normalized to lumine-
and for normalization calculations. Samples were measured in scence values of medium only. All values were calculated in
triplicate. A sample containing Milli-Q water was run as nega- relation to the caspase-3/7 activity of the untreated cells.
tive control. Cq values were automatically calculated with the
COX inhibition assay. The inhibition of isolated ovine
Lightcycler®96 software (Roche Diagnostics). Concentration COX-1 and human recombinant COX-2 was determined at
and purity of RNA and cDNA samples, respectively, were deter- 10 µM and/or 100 µM of the respective compounds by enzyme
mined with a NanoDrop 2000 UV-vis spectrophotometer immunoassay (EIA) (COX Inhibitor Screening Assay, Cayman
(Thermo Fisher, Waltham, USA). COX-1 and COX-2 expression Chemicals, Ann Arbor, USA) according to the manufacturer’s
was absolutely quantified with a calibration curve using COX-1 protocol. Isoenzymes were incubated with the respective com-
and COX-2 templates (Origene, Rockville, USA) at 0–107 copies pound for exactly 10 min. Results were calculated as the mean
per well. Both sample values and calibration curve values were of duplicate determination and represent the mean SD of ≥3
normalized to the Cq value of β-actin of the respective cell line. independent experiments. Untreated control was set to 0%
Determination of cell biomass with crystal violet staining. inhibition.
The in vitro testing of the new compounds for their effects on
Determination of inhibition of cellular PGE2 synthesis.
cell biomass was carried out according to a modified protocol HT-29 cells were seeded in 24-well plates (5 × 104 cells per well,
previously described.28 Cells were seeded in their exponential 500 µL per well) and incubated at 37 °C for 24 h. After addition
growth phase in complete DMEM in 96-well microtiter plates of compounds at a concentration of 10 µM, cells were incu-
(2 × 103 cells per well, 100 µL per well) in quadruples. Plates bated for another 24 h at 37 °C in a 5% CO2/95% air atmo-
were kept at 37 °C for 24 h prior to addition of complete sphere. Arachidonic acid (50 µM, Sigma-Aldrich, St Louis, USA)
medium containing the vehicle, the positive controls Cisplatin was added to the cells and COX-catalyzed enzymatic turnover
and Co-ASS, and the newly synthesized compounds at indi- was stopped 1 h thereafter by removing the supernatant
cated concentrations, respectively. After an incubation time of medium. PGE2 concentration in the supernatant was
72 h, medium was aspirated, cells were washed with PBS and measured by EIA (Prostaglandin E2 Kit – Monoclonal, Cayman
fixed with a solution of 1% (v/v) glutaric dialdehyde in PBS. Chemicals) according to the manufacturer’s protocol. Results
Cell biomass was determined via staining of the chromatin of were calculated as the mean of duplicate determination and
adherent cells with crystal violet, extraction of the stain with represent the mean
ethanol (70%) and subsequent measurement of absorbance at Untreated control was set to 0% inhibition of PGE2 synthesis.
590 nm. Mean values SD of ≥4 independent experiments Cellular uptake studies. To assess the cellular uptake of the
SD of 3 independent experiments.
were calculated and the effects of the compounds were alkyne complexes, the cobalt content in HT-29 colon and
expressed as percentage of the cell mass of a vehicle-treated MCF-7 as well as MDA-MB-231 breast carcinoma cells was
control, which was set at 100%. The IC50 values were calcu- quantified by high-resolution continuum-source atomic
lated with Prism 7.0 (GraphPad, San Diego, CA) using non- absorption spectrometry (HR CS AAS). The cobalt levels were
linear regression and the decadal logarithm of the inhibitor correlated to the protein contents which were measured by
versus variable slope response equation. The top constraint application of the Bradford’s method.31
was set to 100%.
Cellular uptake studies. Exponentially growing HT-29, MCF-7,
Determination of metabolic activity. Exponentially growing and MDA-MB-231 cells, respectively, were seeded in 6-well
cells were seeded in 96-well plates (HT-29, MDA-MB-231, tissue culture plates in 2 mL of completed DMEM and incu-
MCF-7: 104 cells per well, 50 µL per well; HS-5: 2 × 104 cells per bated at 37 °C in a 5% CO2/95% air atmosphere. The medium
well, 50 µL per well) in triplicates. Appropriate concentrations was aspirated after 24 h and completed DMEM containing the
of compounds were added 3 h (HT-5: 1 h) thereafter. After a respective drug (10 µM) or the vehicle DMSO was added. After
three-day culture at 37 °C in a 5% CO2/95% air atmosphere, the appropriate incubation periods (1 h, 2 h, 4 h, 6 h, 24 h),
cultures were analyzed for metabolic activity using a modified the medium was removed and the cells were detached by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide washing once with PBS followed addition of accutase (GE
(MTT) assay (EZ4U kit; Biomedica, Vienna, Austria) according Healthcare BioSciences, Pasching, Austria) and harvested by
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collecting them with PBS. The cell suspension was centrifuged
(8000 rcf, 3 min, RT) and the supernatant was discarded. The
isolated cell pellet was washed with PBS and stored at −20 °C
until further analysis. Immediately after thawing, the cell
pellet was resuspended in twice distilled water and lysed by
sonification (20 s, nine cycles, 80–85% power). The cell lysate
was appropriately diluted with Triton-X-100 (final concen-
tration: 0.1%) and stabilized with HNO3 13% (final concen-
tration: 10%) to be analyzed for cobalt content by HR CS AAS.
The cellular concentration of the compounds is expressed as
the amount of compound (pmol) related to cellular protein
mass (µg). Values were calculated as the mean of 3 indepen-
dent experiments.
HR CS AAS. For the cobalt quantification, a contrAA
700 high-resolution continuum-source atomic absorption
spectrometer (Analytik Jena AG) was employed. Pure samples
of the respective complexes were utilized as standards and cali-
bration was conducted in a matrix matched manner. The
mean integrated absorbances of triplicate injections were used
throughout the studies. Samples were directly injected into
coated standard graphite tubes (Analytik Jena AG). The time-
temperature furnace program applied for the determination of
cobalt was optimized regarding pyrolysis and atomization
temperatures. It can be found as ESI (Table S2†). Cobalt was
quantified at a wavelength of 240.7254 nm.
PG
qPCR
SD
SE
THF
Prostaglandin
Quantitative real time polymerase chain reaction
Standard deviation
Standard error
Tetrahydrofurane.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank Philipp Schuster and Sina Katharina Götzfried
(University of Innsbruck, Department of Pharmaceutical
Chemistry) for resynthesizing the compounds and for perform-
ing the HPLC analysis, Peter Enoh (University of Innsbruck,
Department of Pharmaceutical Chemistry) for carrying out the
high-resolution mass spectrometry and Caroline Gallati
(University of Innsbruck, Department of Pharmaceutical
Chemistry) for measuring the 1H-NMR spectra.
Quantification of protein content. The protein content of cell
lysates was quantified by the method of Bradford.31 In brief,
the lysates were appropriately diluted with twice distilled
water. To each 20 µL thereof 200 µL of Bradford reagent were
added respectively in 96-well plates. After incubation for 5 min
at RT, the absorbances were read at 595 nm in a microplate
reader (Thermo Scientific Multiskan GO). Solutions of
albumin bovine fraction V diluted with twice distilled water
(31.25–1500 μg mL−1) were used as calibration standards.
Samples were measured in duplicate.
Statistical analysis. The Wilcoxon rank sum test was used to
analyze the significance of differences between cell biomass,
proliferation, metabolic activity, and apoptosis induction in
the absence and the presence of a variable concentration of
the test compounds (NCSS software, Kaysville, UT, USA).
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